Method and test kit for analysis of histamine receptor sites of mammalian cells

ABSTRACT

A method and test kit for selectively staining histamine or histamine blocker sites on mammalian cells is described. Novel fluorescently labeled histamines or histamine blocker compounds are described. The method allows the visualization of sites on individual cells which are receptors for the histamines or histamine blockers.

This is a divisional of co-pending application Ser. No. 06/871,098 filedon June 5, 1986, now U.S. Pat. No. 4,830,961.

BACKGROUND OF THE INVENTION

(1) Summary of the Invention

The present invention relates to a method and test kit for analyzinghistamine receptor sites of mammalian cells using novel fluorescenthistamine or histamine blocker compounds which bind to the sites. Inparticular, the present invention relates to the use of 9-phenylxanthenefluorescent labeled histamine compounds for the analysis.

(2) Prior Art

The principal prior art is described in U.S. Pat. No. 4,474,876 toOsband et al. This patent describes the binding of multiple histamine orhistamine blockers to a protein or peptide molecule with fluorescentcompounds bonded to the protein or peptide for analytical purposes. Theproblems is that this fluorescent molecule does not bind to the sites ofthe cells or cell fragments in a way which allows histamine bindingsites of the cells to be distinguished. A paper published by Muirhead etal in the Journal of Immunology 135, 4120 to 4128 (1985) suggests thatthis method is not effective to label only histamine active sites of thecells.

Wulf et al in Proceedings of National Academy of Science 76 (4498-4502)1979 describe the fluorescent labeling of phallotoxin. U.S. Pat. No.4,300,310 to Galbraith describes fluorescent labeling of heterokaryons.U.S. Pat. No. 4,435,509 to Berthold et al describes fluoresceinisothiocyanate labeling which is enhanced by hypochlorite. None of thisprior art shows site selective fluorescent labeling of cells.

OBJECTS

It is therefore an object of the present invention to provide animproved method and test kit for staining mammalian cells or cellfragments thereof using novel directly fluorescently labeled histamineor histamine blocker compounds. Further it is an object of the presentinvention to provide a staining method which is inexpensive andreliable. These and other objects will become increasingly apparent byreference to the following specification and the Figures.

IN THE FIGURES

FIG. 1 shows binding of the preferred F-His compound of the presentinvention to adherent human polyphosphonuclear leucocytes (PMNs) inHank's balanced salt solution. Samples were treated as described in thespecification with F-His at 10⁻⁵ M followed by three washes with abuffer solution. Bright-field (a, c, e, g, i, k, and m) and fluorescence(b, d, f, h, j, 1, and n) images were recorded. These photomicrographsshow (a, b) stationary PMN; (c, d) PMN with excess histamine; (e, f) PMNwith cimetidine; (g, h) PMN during chemokinesis; (i, j) PMNspontaneously polarized; (k, l) PMN polarized during chemotaxis andfixed with 2% paraformaldehyde; (m,n) PMN polarized during chemotaxiswithout fixation; (o,p) PMN polarized with f-met-leu-phe in the presenceof ethylene diamine tetramine (EDTA) to inhibit locomotion. The areas ofthe uropods are indicated with arrows. (a,b×2,400; c-p×1,200).

GENERAL DESCRIPTION

The present invention relates to an improved method for analyzingmammalian cells to determine the receptors for histamine by preparingcells or cell fragments for staining and then staining the cells with afluorescent labeled histamine or histamine blocker, which comprisesstaining the cells with a compound of the formula: ##STR1## whereinFLUOR is a fluorescent group directly bonded to the ##STR2## group andwherein ##STR3## is selected from histaminic and histamine blockergroups which bond to histamine sites on the cells to therebyfluorescently stain the cells selectively at the histamine receptors.

The present invention also relates to a kit for assaying histaminereceptors on cells which comprises a compound of the formula ##STR4##wherein FLUOR is a fluorescent group directly bonded to the ##STR5##group and wherein ##STR6## is a histamine or histamine blocker group andwherein the compound selectively binds to histamine receptors ofmammalian cells wherein the compound is in a prepackaged form for theassaying.

The present invention further relates to a preferred compound of theformula: ##STR7## wherein FLUOR is a fluorescent group bonded to the NHgroup and wherein ##STR8## is a histamine group or histamine blockergroup and wherein the compound binds to histamine sites of mammaliancells.

The fluorescent compounds which can be attached to the histamine orhistamine blocker molecule are well known to those skilled in the art.Fluorescent molecules with an isothiocyanate group are preferred sincethey easily react with the amino group on the histamine or histamineblocker compound. The useful fluorescent compounds include eosin,erythrosin, rodamine, Texas red, nitrobenzoxadiazole, coumain, acridine,stilbene, pyrene and antnracene compounds. These compounds generallyhave fused rings and various linking groups which react with aminogroups, including isothiocyanate, succinimidyl, sulfonyl halide (iodo,or chloro), and acid groups. All of these fluorescent molecules aredescribed in the Handbook of Fluorescent Probes and Research Chemicals,and available from Molecular Probes, Junction City, Oreg. and are wellknown to those skilled in the art.

The xanthene dye derived compounds as the fluorescent compounds arepreferred, particularly the 9-phenylxanthenes. The most preferredcompounds are fluorescein 5-isothiocyanates because of the superiorresults obtained in the method of the present invention.

The preferred FLUOR-Histamine compounds have the formula: ##STR9## andmetal salts thereof. U.S. Pat. No. 4,474,876 identifies many relatedhistamine blocker or agonist compounds which can be labeled with thefluorescent compounds. These include 2(2-pyridyl)ethylamine, dimaprit,substituted histamines including 4-methylhistamine, diphenhydramine andcimetidine.

SPECIFIC DESCRIPTION

In this experimental effort, a univalent and bioactive fluorescentderivative of histamine was bound to the surface of humanpolymorphonuclear (PMN) leukocytes. Free histamine was found to competewith this derivative for binding sites. Histamine H2 receptorspecificity was indicated by binding inhibition experiments usingcimetidine (H2-specific) but not diphenhydramine (H1-specific). Videointensification fluorescence microscopy was used to determine thedistribution of histamine receptors in living PMNs. Receptors appear asrandomly distributed clusters upon stationary cells. During randomlocomotion, receptors were restricted to the ends of pseudopods whilechemotaxis leads to receptor localization at lamellipodia and uropods.Ligand-receptor complexes were restricted to the cell surface as shownby quenching exterior fluorescence with crystal violet. Thereforepinocytic uptake cannot account for the observed receptor localizationor clustering. As a further control, the lipid analog DiI remaineduniformly distributed during all conditions. Histamine-mediatedinhibition of adherence may be related to formation of ligand-receptormembrane domains at adherence sites.

Histamine is a well-known vasoactive amine that is released during theinflammatory component of acute allergic responses. Several laboratorieshave variously reported that histamine modulates polymorphonuclear (PMN)leukocyte chemokinesis, chemotaxis, degranulation, oxidative metabolism,and adherence. The broad spectrum of physiological reactions mediated byhistamine are triggered by cell surface receptors. However, the cellsurface topography of histamine receptors and their modulation duringdistinct cellular activities are not known. Furthermore, there was nosuitable ligand available to obtain this information.

A univalent and bioactive fluorescent derivative of histamine that bindswith high specificity to the surface of living human PMNs wassynthesized. In addition to providing fresh information regarding cellsurface properties of histamine receptors with a new fluorescence tool,the studies have suggested a possible structure-function correlationsince the histamine-fluorescein conjugate (F-His), which inhibitsadherence, accumulates at sites generally associated with adherenceactivity.

MATERIALS AND METHODS

Preparation of F-His. To prepare F-His, the side-chain nitrogen wasconverted to a secondary amine using fluorescein isothiocyanate (FITC)in ethanoloic NaOH. 14 mg of histamine and 10 mg of FITC were dissolvedin 1:1 (vol./vol.) ethanol:0.1M NaOH and allowed to react in alight-tight test tube for 48 hours at 4° C. Histamine and FITC alonewere treated in the same fashion as controls. Samples were acidifiedfollowed by thin-layer chromatography using 3:1:1 of chloroform,methanol, and ethanol as developing solvent. Analysis of thechromatograms revealed R_(f) values of 0.45, 0.55, and 0.81 forhistamine, F-His, and FITC, respectively.

Preparation of Cells. PMNs were obtained from clot preparations asdescribed (Petty, H. R., et al., Biophys. J. 47, 837-840 (1985);Zigmond, S. H. J. Cell Biol. 75, 606-616 (1977)). This preparatorymethod was chosen because it minimizes perturbation of cell functioncaused by handling and purification procedures. Drops of fresh bloodwere placed on glass coverslips in a humidified atmosphere at 37° C. for30 minutes followed by gentle rinsing with saline to remove the clot.These preparations contained 92 to 95% neutrophils, ≦3% eosinophils, and4-8% monocytes. Adherent PMNs were employed for further experimentation.

Cell Adhesion. Cell adhesion was measured by the method of Keller et al(Keller, H. U., et al., Exp. Cell. Res. 122, 351-362 (1979)).Neutrophils were isolated from human peripheral blood according to themethod of Ferrante & Thong (Ferrante, A., and Y. H. Thong, J. Immunol.Meth. 36, 109-117 (1980)). The Ficoll-Hypaque solution was obtained fromPackard Inst. Co. (Downers Grove, Ill. Neutrophils at 10⁶ /ml in Hank'sbalanced salt solution (HBSS; Gibco, Grand Island, N.Y.) were incubatedin tissue culture chamber/slides for 15 or 30 min. at 37° C. The slideswere thoroughly washed, fixed with ethanol, then stained with Giemsa.Cell counts per unit area were made in the central region of the slide.Cells were untreated or treated with histamine or F-His. Adherence isgiven as % inhibition in comparison to matched controls.

Chemotaxis. Chemotaxis chambers were constructed as described by Zigmond(Zigmond, S. H., J. Cell Biol. 75, 606-616 (1977)).N-formylmethionylleucylphenylalanine at 1 μM in HBSS+2% heat-inactivatedfetal calf serum was used. In some cases coverslips were removed fromthe chamber followed by fixation with 2% paraformaldehyde in saline for3 to 5 minutes. Samples were then labeled as described below. Forstudies with living cells, receptors were labeled as described belowfollowed by insertion into the Zigmond chamber. In all cases the chamberwas kept at a nominal temperature of 37° C. using an Incu-stageincubator (Lab-Line Inst., Melrose Park, Ill.).

Cell Labeling. Cells were labeled with F-His or1,1'-dioctadecyl-3,3,3'3'-tetramethylindicarbocyanine (diI) oncoverslips. F-His at 25 μM in 4:6 ethanol: H₂ O was diluted in PBS tovarious final concentrations prior to application to coverslips. Intypical experiments 10 μM F-His was used to label cells for 5 minutes at4° C. The reagent diI was obtained from Molecular Probes, Inc. (JunctionCity, Oreg.). For diI labeling 10 μl of a 300 μM solution in ethanol wasdiluted into 1 ml of PBS. Labeling was performed for 5 minutes at roomtemperature. Samples were washed three times with PBS.

Fluorescence Microscopy. Cells were examined in a Zeiss fluorescencemicroscope equipped with special excitation and detectioninstrumentation. The device is a combination of fluorescence recoveryafter photobleaching and video-intensified microscopy (Petty, H. R., etal., Proc. Natl. Acad. Sci. 77 6587-6591; Willingham, M. C., et al. Cell13, 501-507 (1978)). An argon-ion laser (model 164-09; Spectra-PhysicsInc., Mountain View, Calif.) operating at 488 nm or 514 nm was used forexcitation of F-His or diI, respectively. The intensity was adjusted byplasma tube current density, neutral density filters, and abeam-splitter assembly (Petty, H. R., et al., Proc. Natl. Acad. Sci. 77,6587-6591 (1980)). The beam was spatially filtered, expanded (EarlingCorp. South Natick, Mass.), and reflected into the epi-fluorescenceport. A light level was chosen which did not photobleach the sample.Leitz 50× (n.a.=1.0) or 100× (n.a.=1.2) water immersion objective wereemployed. In these experiments, the image was reflected onto an RCAsilicon-intensifier tube held in a Dage-MTI model 65 camera. Videosignals were recorded on a Panasonic NV-8050-high resolution videorecorder and displayed on an Audiotronics monitor. The photographsreported were taken from the screen using a Polaroid camera.

RESULTS

The bioactivity of F-His was tested using a previously describedadherence assay (Keller, H. U., et al., Exp. Cell. Res. 122, 351-362(1979)). These experiments showed 55±7% adherence of PMNs to glasscoverslips in comparison to controls in a 15 min. assay at 10⁻⁵ Mhistamine and F-His, respectively.

After exposure of PMNs to F-His at 4° C. for 15 minutes at 10⁻⁵ M,fluorescence was distributed in clusters (FIGS. 1a and b). Maximalbinding of F-His to PMNs was found at 10⁻⁵ M. This is in good agreementwith the results of Osband et al (Osband, M. E., et al., Blood 58, 87-90(1981)) using radiolabeled histamine. The clusters were predominantlyassociated with the cell surface as judged by moving the focal planethrough the cell (for additional controls, see below). The clusterscannot be due to extracellular crosslinkage since F-His is univalent.Binding was specific since inclusion of a 100-fold excess of histamineabrogated binding (FIGS. 1c and d). In addition, this controls forbulk-phase pinocytic uptake {but not receptor-mediated uptake; seebelow} of F-His and non-specific membrane permeation. Both of thesephenomena were undetectable. We have also tested the effects of the H2antagonist cimetidine on F-His binding to PMNs. In FIGS. 1e and f weshow the PMNs labeled with 10⁻⁵ M F-His in the presence of 1 mMcimetidine; no labeling can be observed. The H1 antagonistdiphenhydramine has no observable effect upon F-His binding at 1 mM(data not shown). These results are in agreement with previous reports(Beer, D. J., et al. Adv. Immunol 35, 209-268 (1984)) indicating the H2specificity of the PMN histamine receptor.

Cell locomotion and polarization result in profound alterations in thedistribution of histamine receptors at the PMN surface. In FIGS. 1g andh we show a PMN undergoing chemokinesis in the presence of F-His.Ligand-receptor complexes were found at the ends of pseudopods andmicrovilli. Fluorescence cannot be found in association with the cellbody. Under these conditions some PMNs spontaneously polarize (FIGS. 1iand j). In this case, receptors were associated with the leading edgeand uropod. Polarization and chemotaxis were induced by gradients off-met-leu-phe (Zigmond, S. H., J. Cell Biol. 75, 606-616 (1977)).Fluorescence was associated with the lamellipodium and/or uropod (FIGS.1k and l) after fixation with 2% paraformaldehyde for 3 to 5 minutes. Inthe absence of fixation, receptors are rapidly redistributed to theuropod (FIGS. 1m and n). In the presence of EDTA, PMN locomotion but notpolarization is inhibited. Again, fluorescence was found at thelamellipodium and/or uropod (FIGS. 1o and p). Small membranous vesiclesarising from retraction fibers at the uropod were also labeled (FIG.1p).

DISCUSSION

The results of the present invention are of technological and biologicalsignificance. F-His represents the first fluorescent histamine receptorprobe which retains biological activity and receptor and pharmacologicalspecificity. These conditions have generally not been fulfilled byprevious histamine receptor probes using protein carries (Ganellin, C.R., et al., Pharmacology of Histamine Receptors. Wright PSG, London(1982); Beer, D. J., et al., Adv. Immunol 35, 209-268 (1984)).

The physiological role of the histamine ligand-receptor clusters isimmediately suggested by their location. Puctate fluorescence isobserved upon stationary cells. Cell locomotion leads to redistributionof fluorescence to pseudopods or to the lamellipodium and uropod. Thesecellular sites are responsible for adherence of PMNs and fibroblasts tosubstrates. Therefore, accumulation of histamine ligand-receptorcomplexes at these sites may sterically exclude membrane componentsparticipating in adhesion or decrease adhesion through the action of asecond messenger such as cAMP. In either case a structure-functioncorrelation is suggested. Accumulation of histamine receptors at theleading edge of migrating PMN may also increase cell sensitivity to anextracellular mediator such as histamine. Previous studies haveindicated that certain cell surface components undergo topographicreorganizations during cell movement, endocytosis, or capping (Oliver,J. M., et al. Sem. Hematol. 20, 282-304 (1983)). The results of thepresent study are consistent with the Oliver-Berlin wave model ofreceptor redistribution (Oliver, J. M., et al. Sem. Hematol. 20, 282-304(1983)). Moreover, the results clearly indicate that cross-linkage ofreceptors by multivalent ligands is not a requirement for cell surfaceresponses fitting the Oliver-Berlin model, although receptor-receptorcross-linkage is not ruled out. The fluorescence label described aboveis applicable to the study of histamine receptors in their many diversephysiological settings using microscopic and flow cytometric techniques.The development of H1- and H2-specific fluorescent labels should allowfor simultaneous localization of these distinct receptors.

The present invention can be used for:

(1) The fluorescence detection and assay of histamine receptors usingflow cytometry, fluorescence microscopy, and fluorescence spectroscopy.

(2) The characterization of peripheral blood leukocytes including:

(A) analysis of normal and diseased leukocytes.

(B) analysis of leukemic cells.

(C) analysis of immunoenhancement mediated by H2 antagonists.

(D) Enumeration of histamine receptor bearing cells.

(3) Fluorescent 1-methyl and 4-methyl histame derivatives which measureH1 and H2 specific cells.

(4) Determination of H1 and H2 mediated neurotransmission pathways.

(5) Measurement of serum histamine levels with a reverse competitionassay.

(6) The fluorescent histamine label can be used to identify andsubsequently remove histamine receptor positive lymphocytes inimmunosuppressive disorders (e.g., AIDS). Histamine suppresses theimmune response.

(7) In addition to the immunological and neurological applicationssuggested above, histamine receptors are associated with:

gastric secretion,

vascular system,

smooth muscle (alimentary, genitourinary, etc.)

heart,

lung and thus can be analyzed with the method of the present invention.Many additional applications for the fluorescent labeled histamines ofhistamine blockers will occur to those skilled in the art.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

I claim:
 1. A compound of the formula: ##STR10## wherein FLUOR is afluorescent 9-phenyl xanthene group moiety with the 9-phenyl groupbonded to the NH group.
 2. A 9-phenylxanthene compound of the formula:##STR11## and metal salts thereof.
 3. A kit for assaying histaminereceptors on cells which comprises a compound of the formula ##STR12##wherein FLUOR is a fluorescent 9-phenyl xanthene moiety with the9-phenyl group bonded to the NH group wherein the compound selectivelybinds to histamine receptors of mammalian cells and wherein the compoundis in a prepackaged form for the assay.
 4. The kit of claim 3 whereinthe compound is: ##STR13##